Figure 1.
tud1 is epistatic to d1, but additive to d61.
(A) Adult plant morphology of wild type (WT) (Nipponbare), d1-c, tud1-5 and d1-c/tud1. Both tud1-5 and d1-c mutants were derived from Nipponbare. The plant height of tud1 is similar to d1-c/tud1, showing that tud1 is epistatic to d1 in plant height. Bar: 10 cm. (B) The grain morphology of wild type (WT), d1-c, tud1-5 and d1-c/tud1 mutants. The vertical grain lengths of tud1-5, d1-c and d1-c/tud1 were shortened compared to wild type. The vertical grain length of tud1-5 is similar to d1-c/tud1, showing that tud1 is also epistatic to d1 in grain length. (C) The patterns of internode elongation in wild type (WT), d1-c, tud1-5 and d1-c/tud1. d1-c is characterized by a dn-type dwarfism, but tud1 and d1-c/tud1 are of dm-type dwarfism and their second internode elongations are specifically inhibited. Arrows indicate start and end of the second internode. Bar: 10 cm. (D) Plant heights for wild type (WT), tud1-4, d61-2 and tud1/d61, showing that these mutants are additive in the reduction of plant height. Bar: 2 cm.
Figure 2.
tud1 is a pleiotropic dwarf mutant.
(A) Adult plant morphology. Comparison of five allelic mutants of tud1 (tud1-1 to -5) with their corresponding wild rice varieties MH63, ZY, SH163, TTP and Nip, respectively. Bar: 10 cm. (B) Comparison of culm (stem) elongation of the tud1 mutant alleles with their corresponding wild types. According to Takeda (1977), tud1-1, tud1-2 and tud1-5 showed dm-type dwarfism, and tud1-3 and tud1-4 showed dn type. Arrowheads indicate the positions of nodes. Bar: 10 cm. (C) Grain morphology. All of the mutants have shortened grains. The upper panel and the lower panel represent the unhulled and hulled seeds, respectively. Bar: 2 mm. (D) Leaf morphology. The leaf blades of tud1 mutants all are dark-green and rugose.
Figure 3.
Cell numbers are reduced in aerial organs of tud1-2.
(A) Aerial parts of three-week old wild type (WT) (upper-left) and tud1-2 (upper-right). Schematic illustration of the third leaf sheaths is shown in middle bottom. Central parts of the leaf sheaths were cut off and the lengths of motor cells between files of stomata were measured. (B) Panicles and internodes of wild type (WT) and tud1-2. PA, panicle; BRI, basic rachis internode; IN, internode. Internode I, II and III are the first, second and third internodes, respectively. Bar: 10 cm. (C) Lemma and palea of WT and tud1-2. LM: Lemma; PL: Palea. Bar: 2 mm. (D,E) Longitudinal sections of the third leaf sheath from one-month old seedlings of wild type (WT) (D) and tud1-2 (E). Bar: 200 µm. (F,G) Longitudinal sections of the third internode from one-month old seedlings of wild type (WT) (F) and tud1-2 (G). Bar: 200 µm. (H,I) Inner epidermal cells of lemma from one-month old seedlings of wild type (WT) (H) and tud1-2(I) observed by SEM. Bar: 100 µm.
Figure 4.
tud1-2 is unlikely a GA-related mutant.
(A) Seeds of wild type (TUD1) and the dwarf mutant (tud1-2) were germinated on agar plates in the presence (+) or absence (−) of 1 µM GA3 (Left), or 30 µM of PAC (Right), and seedlings were examined 10 days after germination. (B) Seedling lengths (heights) in response to GA3 treatment in the wild type (blue) and tud1-2 (pink). (C) Changes in SLR1 protein levels triggered by GA3 or PAC treatment. Young seedlings of the wild type (WT) and tud1-2 were treated with (+) 1 µM GA3, 30 µM PAC or control solution (−), as described in Figure 4A and 4B, and their extracts subjected to western blot analysis by anti-Tubulin and anti-SLR1 antibodies, respectively. (D) A plate assay for α-amylase induction. Left: wild type (WT); Right: tud1-2. (E) Gross morphology of tud1-2 and a new eui1 mutant in the tud1-2 background. From left to right; wild type (WT), tud1-2, eui1-h,tud1-2/eui1 (right). Bar: 10 cm. (F) Gross morphology of tud1-2, slr1 and tud1-2/slr1 mutants. From left to right; wild type (WT), tud1-2/slr1, tud1-2 and slr1 (right). Bar: 15 cm.
Figure 5.
tud1-2 is a BR insensitive mutant.
(A,B) Photomorphogenic phenotypes of tud1 grown in the dark. Plants of Taizhong 65 (WT), d61-2 (BR-insensitive mutant), wild type (WT) and tud1-2 were grown in complete darkness (A). Arrows indicate nodes and arrowheads. Bar: 2 cm. The lengths of internodes and mesocotyls were measured after ten days growth. Data presented are the means of results from five plants (B). Error Bars = SD. (C,D) Effects of 24-eBL on the degree of inclination of the leaf lamina in wild type (WT) and tud1 plants. Typical responses of the second leaf lamina joint from wild type and tud1-2 plants to 24-eBL at 0 ng, 10 ng, 100 ng or 1000 ng. Bar: 2 cm (E). The dose responses to 24-eBL, for the bending angle, for WT and tud1-2 (F). Data presented are the means of results from a total of five plants. Error Bars = SD. (E,F,G) Relative expression levels of BRD1 (E), OsDWARF4 (F) and D61 (G) in different internodes. WT-I and tud1-2-I represent the uppermost internode, WT-II and tud1-2-II represent the second elongated internode, respectively (from top to bottom).
Figure 6.
TUD1 is a functional E3 ligase.
(A) High resolution linkage and physical map of tud1 locus. Horizontal lines represent chromosome 3 and vertical bars the molecular markers. The numbers of recombinant plants are indicated between the markers. The physical map of the tud1 locus was constructed using 7 BAC clones and the candidate region of the tud1 mutation was found between markers P2 and P3. The genomic structure of the TUD1 gene and the positions of mutations in tud1 alleles are also shown. The black box indicates the single exon. The mutated DNA sequences of tud1 alleles are shown in the bottom. The tud1-1 has one-base insertion near the initiation codon. The tud1-2, tud1-4 and tud1-5 have different one-base substitutions in the coding sequence. The tud1-3 has a one-base substitution and 62 bp base deletion in the exon. CEN:centromere, 3S: the short arm of chromosome 3, 3L: the long arm of chromosome 3. (B) Phenotypic complementation by introduction of the TUD1 gene. Nipponbare was the parent for tud1-2 and used as the wild-type plant. Left, the wild type (Nipponbare); center, transformant-TUD1; right, transformant-control vector. Bar: 10 cm. (C) Schematic structures of TUD1 protein and its mutant alleles. Black bars indicate TUD1 coding regions. Red bars depict U-box motifs. Green bars represent the aberrant truncated protein of tud1-1 and partial amino acids residues of tud1-3 due to a frameshift mutation. Numbers represent the amino acids of protein sequences. (D) Ubiquitination assays with GST-TUD1. GST-TUD1, GST, and positive controls of RMAI and CiP8 were assayed for E3 activity in the presence of E1 (from wheat), E2 (UBCh5b), and 6×His tag ubiquitin (Ub). The numbers at left denote the molecular mass in kilodaltons. Samples were resolved by 8% SDS-PAGE. The nickel-horseradish peroxidase was used to detect His tag ubiquitin. (E) Ubiquitination assays with GST-TUD1 and its mutant variants. GST-TUD1 and its mutant forms (GST-tud1-1, GST-tud1-3 and GST-tud1-4 fusion proteins) were assayed for E3 activity. The reaction conditions were the same as in (D).
Figure 7.
TUD1 is predominantly associated with the plasma membrane and physically interacts with D1.
(A) Expression of the TUD1-GFP fusion protein in rice protoplasts. The 35S:sGFP and 35S:TUD1-sGFP constructs were transformed into mesophyll protoplasts prepared from rice seedlings and the expression of the introduced genes was viewed after 16 h by confocal microscopy under dark-field or light-field conditions. Bar: 10 µm. (B) BiFC detection of the TUD1-D1 interaction in rice protoplasts. CFP: cyan fluorescence protein. Bar: 5 µm. (C) Interaction between TUD1 and D1 detected by yeast two-hybrid assays. The full-length D1 and TUD1 cDNAs were cloned into pGADT7 and pGBKT7, respectively. Yeast AH109 cells were transformed with the vectors indicated and are shown after three days on selective medium. “+” and “−” represent positive and negative control, respectively. D1 or TUD1 alone shows weak self-activation. TUD1+D1 show yeast cells transformed with pGBKT-TUD1 and pGADT7-D1 together. (D) Pull-down assay for TUD1−D1 interaction. Purified D1 fusion protein was incubated in buffer with either GDP or GTPγS or blank (control) for 2 hours before adding the GST-TUD1 to the binding assay buffer, then precipitated with glutathione-agarose beads. The precipitates were separated by gel electrophoresis and probed with anti-His or anti-GST, respectively.